1. Field of the Invention
The present invention concerns the processing of biological mixtures through extraction of those mixtures with organic alcohols and/or halogenated hydrocarbons in order to remove exogenous or endogenous lipid soluble chemicals, for example, chemicals used in the inactivation of viruses. The present invention also concerns protein-containing blood products having an extent of inactivation of virus greater than 6 logs for the virus and having a retention of functional activity for particular biological blood proteins of at least 45%.
2. Background Information
Blood can contain each of several different viruses including but not limited to hepatitis B virus (HBV), non-A, non-B hepatitis virus (NANBHV), cytomegalovirus, and immunodeficiency viruses. It is highly desirable to inactivate these viruses in the course of preparing vaccines and prior to the therapeutic application of blood and blood fractions. Both physical (e.g., heat, irradiation) and chemical (e.g., aldehydes, organic solvents, detergents, etc.) methods have been used to inactivate viruses such as HBV in mammalian blood and blood fractions. Inactivation renders a virus non-infectious and non-pathogenic.
Numerous attempts have been made to inactivate viruses such as hepatitis B virus (HBV) in mammalian, especially human, blood plasma. It is the practice in some countries to effect inactivation of the hepatitis B virus in the blood plasma by bringing the plasma into contact with a viral inactivating agent which crosslinks the proteinaceous portions of hepatitis B virus or which interacts with the nucleic acid of the virus. For instance, it is known that hepatitis B virus is inactivated by contact with an aldehyde, such as formaldehyde.
Procedures that have been tested for inactivating viruses in protein biologics include the following:
(1) neutralization with specific antibodies (E. Tabor, D. L. Aronson, R. J. Gerety, "Removal of Hepatitis B Virus Infectivity from Factor IX Complex by Hepatitis B Immune Globulin", Lancet, (1980), 2, 68-70; H. G. J. Brummelhuis, J. Over, L. A. Duivis-Vorst et al, "Contributions to the Optimal Use of Human Blood. IX&gt;Elimination of Hepatitis B Transmission by (Potentially) Infectious Plasma Derivatives", Vox San, (1983), 45, 205-216),
(2) ultraviolet irradiation ("UV") (J. W. Oliphant, A. Hollaender, "Homologous Serum Jaundice Experimental Inactivation of Etiologic Agent in Serum by Ultraviolet Irradiation", Public Health Rep., (1945), 61, 598-602; F. O. MacCallum, "Homologous Serum Hepatitis", Proc. Roy. Soc. Med., (1946), 39, 655; R. Murray, J. W. Oliphant, J. T. Tripp et al, "Effect of Ultraviolet Radiation on the Infectivity of Icterogenic Plasma, JAMA, (1955), 157, 8-14),
(3) beta-propiolactone ("BPL") and ultraviolet (UV) radiation (F. W. Hartman, G. A. LoGrippo, "Combined beta-Propiolactone and Ultraviolet Irradiation for Plasma Sterilization", F. W. Hartman, G. A. LoGrippo, J. G. Mateer et al, eds. Hepatitis Frontiers. Henry Ford Hosp. International Symposium, Boston, Little, Brown & Co., (1957), 407-416; R. Kotitschke, W. Stephan, "Kominierte Behandlung von Gerinnungsfaktoren in Humanplasma mit .beta.-Propiolacton and UV. Struktur und Funktion des Fibrinogens", H. Schroeer, G. Hauck, F. Zimmerman et al, eds., Blutgerinnung und Mikrozirkulation Stuttgart: Verlag, (1976), 222-228; G. A. LoGrippo, H. Hayashi, "Efficacy of beta-Propiolactone with Ultraviolet Irradiation of Hepatitis B Antigen in Human Plasma Pools, Henry Ford Hosp. Med. J., (1973), 21, 181-186; D. Heinrich, H. Berthold, "Application of Cold Sterilized Prothrombin Complex Concentrates in Man: Clinical and Serological Studies", The 13th International Congress of the World Federation of Hemophilia, Tel Aviv, July 8-13, 1979; W. Stephan, A. M. Prince, "Efficacy of Combined Treatment of Factor IX Complex (PPSB) with .beta.-Propiolactone (b-PL) and Ultraviolet (UV) Irradiation", Haemostasis, (1981), 10, 67; A. M. Prince, W. Stephan, B. Brotman, ".beta.-Propiolactone/Ultraviolet Irradiation: A Review of its Effectiveness for Inactivation of Viruses in Blood Derivatives", Rev. Infect. Dis., (1983), 5, 92-107; W. Stephan, A. M. Prince and R. Kotitschke, "Factor VIII Concentrate from Cold Sterilized Human Plasma", Develop Biol. Stand, (1983), 54, 491);
(4) heat applied to the product in the liquid state (S. S. Gellis, J. R. Neefe, J. Stokes Jr. et al, "Chemical, Clinical and Immunological Studies on the Products of Human Plasma Fractionation, XXXVI Inactivation of the Virus of Homologous Serum Hepatitis in Solutions of Normal Human Serum Albumin by Means of Heat", J. Clin. Invest., (1948), 27, 239-244; R. Murray, W. C. L. Diefenbach, "Effect of Heat on the Agent of Homologous Serum Hepatitis", Proc. Soc. Exp. Biol. Med., (1953), 84, 230-231; J. P. Soulier, C. Blatix, A. M. Courouce et al, "Prevention of Virus B Hepatitis (SH Hepatitis)", Am. J. Dis. Chid., (1972), 123, 429-434; T. Shikata, T. Karasawa, K. Abe et al, "Incomplete Inactivation of Hepatitis B Virus After Heat-Treatment at 60.degree. C. for 10 hours", J. Infect. Dis., (1978), 138, 242-244; E. Tabor, R. J. Gerety, "The Chimpanzee Animal Model for non-A non-B Hepatitis: New Applications", W. Szmuness, H. J. Alter, J. E. Maynard eds. Viral Hepatitis: 1981 International Symposium., Philadelphia: The Franklin Institute Press, (1981), 305-317; Von. N. Heimburger, H. Schwinn, P. Gratz et al, "Faktor VIII-Konzentrate, Hochgereinigt und in Losung Erhitzt, Arzneim-Thromb/Drug Res., (1981), 31, 619; Tabor, G. Murano, P. Snoy et al, "Inactivation of Hepatitis B Virus by Heat in Antithrombin III Stabilized with Citrate", Thromb. Res., (1981), 22, 233-238; D. Menache, D. L. Aronson, "Measures to Inactivate Viral Contaminants of Pooled Plasma Products, R. Y. Dodd, L. F. Baker eds. Infection Immunity and Blood Transfusion Proc. XVII Annual Scientific Symposium, May 9-11, 1984. New York, Alan R. Loss, (1985), 407-423), or in the dry state (G. Dolana, D. Tse, W. Thomas et al, "Hepatitis Risk Reduction in Hemophilia: A Heated Factor VIII Preparation", J. Amer. Soc. Hematol., (1982), 60, (Suppl 1), 2102; F. R. Hollinger, G. Dolana, W. Thomas et al, "Reduction of Infectivity of Hepatitis B. Virus (HBV) and a non-A, non-B Hepatitis Agent by Heat Treatment of Human Antihemophilic Factor (AHF) Concentrates", L. R. Overby, F. Deinhardt, J. Deinhardt, eds., Viral Hepatitis: Second International Max von Pettenkofer Symposium, New York: Marcel Dekker, Inc., (1983) 245-246; F. B. Hollinger, G. Dolana, W. Thomas et al, "Reduction in Risk of Hepatitis Transmission by Heat-Treatment of a Human Factor VIII Concentrate", J. Infect. Dis., (1984), 150, 250-262),
(5) lipid solvents with the addition of surface active agents (A. M. Prince, B. Horowitz, B. Brotman et al, "Inactivation of Hepatitis B and Hutchinson Strain non-A, non-B Hepatitis Viruses by Exposure to Tween 80 and Ether", Vox Sang, (1984), 46, 36-43; A. M. Prince, B. Horowitz and B. Brotman, "Sterilisation of Hepatitis and HTLV-III Viruses By Exposure to Tri(n-Butyl)Phosphate and Sodium Cholate", The Lancet, 706-710, March 29, 1986), and lipid solvents without the additive of surface active agents (S. M. Feinstone, K. B. Mihalik, T. Kamimura et al, "Inactivation of Hepatitis B Virus and non-A, non-B Hepatitis by Chloroform, Infect. Immunol., (1983), 41, 816-821; D. W. Bradley, J. E. Maynard, H. Popper et al, "Posttransfusion non-A, non-B Hepatitis: Physiochemical Properties of Two Distinct Agents", J. Infect. Dis., (1983), 148, 254-265).
Neutralization by specific antibody is limited by antibody availability (hepatitis B virus only, so far), (Tabor et al, Lancet, (1980), supra and Brummelhuis et al, Vox Sang, (1983), supra) ultraviolet irradiation and thermal inactivation methods have been variably successful (S. S. Gellis, J. R. Neefe, J. Stokes, Jr., L. E. Strong, C. A. Janeway, G. Scatchard, "Chemical, Clinical and Immunological Studies on the Products of Human Plasma Fractionation. XXXVI. Inactivation of the Virus of Homologous Serum Hepatitis in Solutions of Normal Human Serum Albumin by Means of Heat", J. Clin. Invest., (1947), 27, 239-244; N. Heimburger, H. Schwinn, R. Mauler, "Factor VIII Concentrate, Hepatitis-Safe: Progress in the Treatment of Hemophilia A", Die gelben Hefte, (1980), 20, 165-174; M. Colombo, V. Carnelli, C. Gazengel, P. M. Mannucci, G. F. Savidge, K. Schimpf, "Transmission of non-A, non-B Hepatitis by Heat-Treated Factor VIII Concentrate", Lancet, (1985), July 1-4; F. E. Preston, C. R. M. Hay, M. S. Dewar, M. Greaves, D. R. Triger, "Non-A, non-B Hepatitis and Heat Treated Factor VIII Concentrates", Lancet, (1985), July, 213; C. Rouzioux. S. Chamaret, L. Montagnier, V. Carnelli, G. Rolland, P. M. Mannucci, "Absence of Antibodies to AIDS Virus in Haemophiliacs Treated with Heat-Treated Factor VIII Concentrate, Lancet, (1985), February, 271-272; P. B. A. Kernoff, E. J. Miller, G. F. Savidge, S. J. Machin, M. S. Dewar, F. E. Preston, "Wet Heating for Safer Factor VIII Concentrate?" Lancet, 1985, September, 721), and beta-propiolactone chemically alters proteins and its carcinogenic properties constitute a hazard to personnel handling it.
U.S. Pat. Nos. 4,481,189 and 4,540,573, the entire contents of which are incorporated by reference herein, describe the use of organic solvent/detergent pairs to reduce by several orders of magnitude the infectivity of hepatitis viruses and certain other viruses contained in plasma and plasma products or added thereto.
Solvent/detergent treatment under appropriate conditions of temperature and contact time effectively disassembles viruses that have envelope proteins associated with lipid, while having negligible effect on the molecular conformations and biological activities of sensitive blood plasma proteins.
The independent effects of organic solvents and detergents in disassembling and attenuating viruses can be facilitated by the presence of both. Removal of detergents, as well as organic solvents, from biological products may be necessary, especially if a particular detergent is not well tolerated by humans or whatever biological system within which the product is to be used.
Other examples of virus inactivation reagents applied to blood include merocyanine, beta-propiolactone and cis-platin.
Other methods used to achieve removal of lipid/detergent mixed micelles from membrane protein complexes may be applicable to removal of the same from plasma products and other biologic products. These have been based on differences in size, buoyant density, charge, binding affinity, phase partitioning and solvent partitioning (A. Helenius and K. Sinous, "Solubilization of Membranes by Detergents", Biochem. Biophys. ACTA, (1975), 415, 29-79).
In the instance of whole blood plasma, blood serum, cryodepleted plasma or cryoprecipitate for direct use in transfusion, implementation of virus sterilization techniques has not been heretofore reduced to practice. Methods of heat sterilization result in unwanted protein denaturation. Immune neutralization is limited, at this time, to hepatitis B virus. Beta-propiolactone/UV treatment results in the destruction of sensitive coagulation factors, e.g., anti-hemophilic factor. Organic solvent/detergent mixtures require implementation of expensive and inconvenient fractionation steps to remove the reagents which cannot be applied conveniently to single units of plasma intended for transfusion or to plasma pools.
Another difficulty in preparing virus sterilized plasma is in filtering the plasma following treatment to maintain bacterial sterility without loss of fibrinogen, antihemophilic factor or other labile proteins.
Thus, because virus sterilization techniques have not been applied to whole blood plasma, serum, cryoprecipitate, cryopoor plasma, virus infectivity upon infusion remains, estimated at 0.05% for hepatitis B and 3% for non-A, non-B hepatitis transmission.
Exogenous chemicals are frequently added to biological mixtures to stimulate synthesis, inactivate viruses contained therein and to stabilize or purify desired components present in the mixture. It is generally desirable to remove these chemicals without otherwise affecting the structure and function of the desired components. For example, the synthesis of certain desired biological products can be induced or enhanced in cell cultures by introduction of phorbol esters into the culture fluid. For example, mezerein may be used to induce gamma interferon production by cultured leukocytes (Y. K. Yip, R. H. L. Pang, J. O. Oppenheim, M. S. Nashbar, D. Henriksen, T. Zerebeckyj-Eckhardt, J. Vilcek, "Stimulation of Human Gamma Interferon Production by Diterpene Esters", Infect. and Immun., (1981) 131-139) or to augment secretion of tumor necrosis factor by cells that produce it (B. D. Williamson, E. A. Carswell, B. Y. Rubin, J. S. Prendergast, H. J. Old, "Human Tumor Necrosis Factor Produced by Human B-cells Lines: Synergistic Cytoxic Interaction with Human Interferon", Proc. Natl. Acad. Sci., USA, (1981), 80, 5397-5401).
Before use in man, phorbol esters must be removed from lymphokine preparations because of the carcinogenic properties of these compounds. Heretofore, phorbol esters have been removed by precipitation, chromatographic, or molecular exclusion processes, (B. Y. Rubin, S. L. Anderson, S. A. Sullivan, B. D. Williamson, E. A. Carswell, L. J. Old, "Purification and Characterization of a Human Tumor Necrosis Factor from the LukII Cell Line", Proc. Natl. Acad. Sci., USA, (1985), 82, 6637-6641).
Process chemicals are also used to purify biopolymers such as blood proteins. For example, polyethylene glycol 4000 (PEG) is used in the purification of antihemophilic factor and a step or steps must be taken to remove PEG.
Dimethylsulfoxide has been added to red blood cell concentrates to stabilize them, especially against freezing, and diethylhexylphthalate also has been shown to increase the stability of red blood cells to storage.